Embryonic stem (ES) cells have enormous potential as a source of therapeutic tissues, including those of the vascular and hematopoietic lineages. The ability to identify and expand cells capable of developing into functional tissue after transplantation into a recipient will be critical to the success of this type of therapy. Although some diagnostic cell surface markers have been identified, methods to expand their numbers in vitro or in vivo are not well defined. Accordingly, the overall goal of the proposed work is to identify cells that can give rise to both vascular and hematopoietic cells and to test the ability of growth factor receptor transgenes to expand these cells in vitro and in vivo. In particular, we plan to develop an efficient and rapid means to track the hematovascular differentiation from ES cells. This project builds on my previous work in which I demonstrated that a truncated erythropoietin receptor transgene (tEpoR tg) can be used to expand hematopoietic stem cells in vitro and to enhance engraftment of these cells in a murine competitive transplantation assay, without stem cell exhaustion or malignant transformation. In addition, we now have ES cells and mice expressing both wt and mutant M-CSF receptors, which function normally in the absence of exogenous M-CSF, but show some similarities in their hematovascular phenotype to the tEpoR tg cells and mice. We have also recently shown that murine ES cells are able to grow and differentiate normally during the expression of a number of fluorescent protein transgenes. I now propose four specific aims designed to isolate and further characterize ES-derived cells giving rise to both vascular and hematopoietic tissues and to test the ability of growth factor receptor transgenes to expand these cells. l) Specific aim (i) is to develop murine ES cells that will express different fluorescent marker proteins upon their progressive differentiation through hemangioblast cells into hematopoietic and endothelial cells. 2) Specific aim (ii) is to test the effects of various transgenes to enhance the development and/or expansion of ES cellderived, hemangioblast, endothelial and/or hematopoietic stem cells. 3) Specific aim (iii) is to isolate enriched cell populations after partial differentiation in vitro to compare gene expression and biologic phenotype to those of other known stem cell populations. 4) Specific aim (iv) is test the capacity of ES cell-derived hemangioblast cells to engraft in newborn mice. Thus, these studies will be helpful in further defining the genes that are important in hematovascular development from ES cells and the potential for generating therapeutic cells or tissues from their in vitro differentiation.